CpG island hypermethylation (the abnormal methylation of cytosine in the context of groups of CpG dinucleotides in the promoter regions of genes) is a common mechanism by which tumor suppressor genes are silenced during cancer development. Different cancer types show distinct patterns of DNA hypermethylation. Thus, analysis of sufficient DNA methylation loci in lung cancer samples should yield loci that are commonly methylated in many cancers, as well as loci that are uniquely methylated in lung cancer. The determination of DNA hypermethylation patterns specific for lung cancer would allow accurate diagnosis of the histological subtype of lung cancer, correlation to clinico-pathological data such as response to treatment and survival (leading to the establishment if predictive markers), and finally, in the future, the development of early detection tools through the identification of methylation changes in the serum and sputum of subjects at risk for lung cancer. To date, however, DNA methylation studies of lung cancer have been limited to single gene loci, or small numbers of loci, which are not hypermethylated at a high enough frequency to be of sufficient diagnostic value. For DNA methylation analysis to be applicable to the accurate diagnosis or early detection of lung cancer, a gene or combination of genes with high sensitivity and specificity for the various histological subtypes of lung cancer needs to be identified. Here we propose to screen a panel of 100 genes to identify loci that are hypermethylated in lung cancer or in certain lung cancer subtypes. We will then evaluate individual loci and panels of loci as general lung cancer markers or markers for lung cancer subtypes. Lastly, we will examine the methylation data to identify correlations of methylation patterns with clinico-pathological features and overall survival. The proposed study will yield data that will form the basis for much larger prospective studies to validate the use of the identified methylation markers to study samples (cancer specimens, sputum and serum) from lung cancer patients and subjects at risk for lung cancer, with the goal to develop markers for lung cancer diagnosis, selection of therapy, prediction of outcome, and detection of recurrence.